MASTITIS, OR INFLAMMATION OF THE MAMMARY GLAND DUE TO INFECTION BY BACTERIA, REMAINS THE PREEMINENT PROBLEM PLAGUING THE DAIRY INDUSTRY TODAY. MASTITIS IS THE MOST COSTLY DISEASE AFFECTING DAIRY CATTLE AND IS RESPONSIBLE FOR THE MOST ANTIMICROBIAL USE ON DAIRY FARMS. INFECTIONS IN THE MAMMARY GLAND CAUSE AN INFLAMMATORY REACTION IN THE UDDER, LEADING TO A HIGHER NUMBER OF CELLS IN THE MILK AS MEASURED BY THE BULK TANK SOMATIC CELL COUNT (MOST COMMON MILK-QUALITY METRIC DETERMINING FARMER PAYMENT). DAIRY PRODUCERS CONSTANTLY STRIVE TO REDUCE THEIR BULK TANK SOMATIC CELL COUNT, AS ACHIEVING THIS RESULTS IN RECEIVING QUALITY PREMIUMS. MANY PROGRESSIVE DAIRY FARMS HAVE CONTROLLED CASES OF MAMMARY GLAND INFECTIONS DUE TO CONTAGIOUS BACTERIA (SPREAD COW-TO-COW), THROUGH USE OF BEST PRACTICES FOR BEDDING MANAGEMENT, AND GOOD HYGIENE (PREVENTING INFECTIONS CAUSED BY BACTERIA IN THE ENVIRONMENT). BY IMPLEMENTING MODERN MASTITIS CONTROL PRACTICES MINIMIZING THE EFFECTS OF BACTERIA CONSIDERED TO BE MAJOR PATHOGENS, THE LEADING CONTRIBUTOR TO BULK TANK MILK SOMATIC CELL COUNT ON FARMS WITH GOOD MILK QUALITY IS MAMMARY GLAND INFECTIONS DUE TO NON-AUREUS STAPHYLOCOCCI. THIS GRAM-POSITIVE GROUP OF BACTERIA IS COMPRISED OF A DIVERSE GROUP OF DIFFERENT SPECIES, AND IS THE PREDOMINANT PATHOGEN GROUP CAUSING INTRAMAMMARY INFECTIONS (IMI) IN DAIRY ANIMALS GLOBALLY. INFECTIONS DUE TO NON-AUREUS STAPHYLOCOCCI (NAS) CAUSE AN INCREASE IN THE SOMATIC CELL COUNT IN THE AFFECTED MAMMARY GLAND, ULTIMATELY HURTING THE PROFITABILITY OF DAIRY PRODUCERS. THIS PROPOSAL WILL GENERATE FUNDAMENTAL KNOWLEDGE ABOUT NON-AUREUS STAPHYLOCOCCI, AND BETTER UNDERSTANDING OF THIS GROUP OF BACTERIA WILL LEAD TO MORE EFFECTIVE PREVENTION AND CONTROL MEASURES OF MASTITIS ON DAIRY FARMS. IMPROVING NAS MASTITIS CONTROL PRACTICES ON DAIRY FARMS ULTIMATELY WILL HELP DAIRY PRODUCERS ACHIEVE QUALITY PREMIUMS FOR THEIR PRODUCT, THEREBY IMPROVING THE PROFITABILITY OF DAIRY FARMS A PRODUCING HIGH-QUALITY, NUTRITIOUS FOOD SOURCE.THE FIRST TWO OBJECTIVES OF THIS PROPOSAL WILL USE DATA DESCRIBING THE INFECTION STATUS OF MAMMARY GLANDS OF DAIRY COWS FROM A PREVIOUSLY-COMPLETED STUDY OF TEN ORGANIC DAIRY HERDS IN VERMONT. CULTURE-BASED METHODS WERE USED TO IDENTIFY IF MAMMARY GLANDS OF DAIRY CATTLE WERE HEALTHY OR INFECTED, AND IF INFECTED, IDENTIFYING THE TYPE OF BACTERIA CAUSING THE INFECTION. THE FARMS STUDIED USED TWO DIFFERENT TYPES OF HOUSING FOR THE ANIMALS, AND A FEW DIFFERENT KINDS OF BEDDING MATERIAL. ADDITIONALLY, COWS IN THE STUDY WERE ASSESSED FOR HOW CLEAN THEIR UDDERS WERE. USING THIS INFORMATION, I WILL A COMPARE THE NUMBER AND FREQUENCY OF INTRAMAMMARY INFECTIONS DUE TO THE NON-AUREUS STAPHYLOCOCCI BETWEEN FACILITY TYPES AND BEDDING MATERIALS, AND EXPLORE IF THERE IS ANY ASSOCIATION BETWEEN NAS INTRAMAMMARY INFECTION AND UDDER CLEANLINESS. THE NON-AUREUS STAPHYLOCOCCI BACTERIA CAUSING INTRAMAMMARY INFECTIONS IN THIS DATA SET WERE PREVIOUSLY SUBMITTED FOR IDENTIFICATION DOWN TO THE SPECIES L,EVEL THROUGH ANALYSIS WITH MASS SPECTROSCOPY. WITH THIS LEVEL OF IDENTIFICATION, I WILL BE ABLE TO EXPLORE THE SPECIES-SPECIFIC DISTRIBUTION, NUMBER AND FREQUENCY OF NAS IMI ON THESE 10 ORGANIC DAIRY HERDS IN VERMONT BETWEEN FACILITY TYPES, BEDDING MATERIALS, UDDER CLEANLINESS, AND OTHER POTENTIAL GROUPINGS BASED ON DIFFERENCES IN MILKING PRACTICES AND ASSOCIATED HERD-LEVEL DATA. THE SOMATIC CELL COUNT (MEASURE OF INFLAMMATORY RESPONSE BY THE UDDER) OF MILK FOR EACH QUARTER OF THE MAMMARY GLAND WAS MEASURED FOR EACH COW STUDIED, SO I WILL BE ABLE TO ANALYZE THE SPECIES-SPECIFIC EFFECT OF DIFFERENT NAS BACTERIA ON THE INFLAMMATORY RESPONSE OF THE UDDER. THE THIRD OBJECTIVE OF THIS STUDY WILL FOCUS ON S. CHROMOGENES, THE MOST COMMON NAS SPECIES IDENTIFIED CAUSING INTRAMAMMARY INFECTIONS ON THESE DAIRY FARMS, MOST FREQUENTLY SEEN IN OUR STUDY AS CAUSING PERSISTENT INFECTIONS (LASTING LONGER THAN 30 DAYS). I WILL COMPARE BACTERIAL ISOLATES FROM TWO INFECTION CATEGORIES OF S. CHROMOGENES INTRAMAMMARY INFECTIONS: ISOLATES FROM AN INTRAMAMMARY INFECTION ASSOCIATED WITH A LARGE INFLAMMATORY RESPONSE BY THE UDDER (AS MEASURED BY QUARTER SOMATIC CELL COUNT), AND ISOLATES FROM AN IMI WITH A SMALL OR ABSENT INFLAMMATORY RESPONSE BY THE UDDER. I WILL USE MOLECULAR GENETIC STRAIN-TYPING METHODS TO ENSURE THAT THESE INTRAMAMMARY INFECTIONS ARE INDEED PERSISTENT (CAUSED BY THE SAME BACTERIA OVER THE OBSERVED TIME). FROM AMONG THESE CONFIRMED PERSISTENT S. CHROMOGENES INTRAMAMMARY INFECTIONS, ISOLATES FROM BOTH GROUPS (THOSE CAUSING HIGHER INFLAMMATION AND LOWER INFLAMMATION IN THE UDDER) WILL BE SELECTED FOR WHOLE-GENOME SEQUENCING. WHOLE-GENOME SEQUENCING WILL DESCRIBE THE ENTIRE GENOMIC DNA SEQUENCE OF THESE BACTERIAL ISOLATES, ALLOWING US TO MAKE COMPARISONS AT THE GENETIC LEVEL BETWEEN THESE TWO GROUPS OF BACTERIA. WE WILL LOOK FOR A GENETIC BASIS FOR THE DIFFERENCE IN INFLAMMATORY RESPONSE IN THE UDDER BETWEEN THESE TWO GROUPS OF BACTERIA, BY IDENTIFYING AND COMPARING VIRULENCE FACTORS BETWEEN THEM (GENETIC FACTORS ASSOCIATED WITH A BACTERIA'S ABILITY TO INFECT OR DAMAGE HOST TISSUES).POTENTIAL ASSOCIATIONS BETWEEN INTRAMAMMARY INFECTIONS CAUSED BY NAS AND DIFFERENT RISK FACTORS (I.E., FACILITY TYPE, BEDDING MATERIAL, AND UDDER CLEANLINESS) AS WELL AS DIFFERENCES IN VIRULENCE FACTORS BETWEEN THE TWO GROUPS OF S. CHROMOGENES WILL BE EXPLORED USING LOGISTIC REGRESSION MODELING. IN ORDER TO SHARE THESE RESULTS AND ANALYSES WITH OTHER MASTITIS RESEARCHERS AND MILK QUALITY PROFESSIONALS, WE WILL PUBLISH TWO MANUSCRIPTS IN THE PEER-REVIEWED LITERATURE. THE FIRST WILL REPORT THE NUMBER AND FREQUENCY OF NAS INTRAMAMMARY INFECTIONS, ENVIRONMENTAL OR MANAGEMENT-RELATED RISK FACTORS IDENTIFIED FOR BOTH NAS AS A GROUP AND BY SPECIES ON ORGANIC DAIRIES IN VERMONT. IT WILL ALSO DESCRIBE THE SPECIES-SPECIFIC EFFECT ON QUARTER SOMATIC CELL COUNT. THE SECOND PUBLICATION WILL COMPARE THE GENETIC DIFFERENCES SEEN BETWEEN THE TWO S. CHROMOGENES GROUPS CAUSING DIFFERENT LEV,ELS OF INFLAMMATION IN THE UDDER, WITH A DESCRIPTION OF DIFFERENCES IN VIRULENCE FACTORS FOUND. THE WHOLE-GENOME SEQUENCES FOR THE 50 S. CHROMOGENES ISOLATES, AS WELL AS THE STRAIN TYPE OF S. CHROMOGENES IDENTIFIED IN THE STEP TO CONFIRM PERSISTENCE OF INFECTION, WILL BE PUBLISHED TO AN OPEN-ACCESS, GLOBALLY RECOGNIZED ONLINE DATABASE AVAILABLE ALL RESEARCHERS (NATIONAL CENTER FOR BIOTECHNOLOGY INFORMATION).THE RESEARCH GOALS OF THIS PROPOSAL ARE TO 1) BETTER UNDERSTAND INFECTION DYNAMICS AND RISK FACTORS FOR INTRAMAMMARY INFECTIONS CAUSED BY NON-AUREUS STAPHYLOCOCCI, 2) EXPLORE HOW DIFFERENT SPECIES AMONG NAS MAY BE OF MORE CONCERN FOR UDDER HEALTH, AND 3) IDENTIFY IF GENETIC DIFFERENCES WITHIN THE MOST COMMONLY-FOUND SPECIES, S. CHROMOGENES, HELP EXPLAIN VARIABILITY IN INFLAMMATORY RESPONSE TO INTRAMAMMARY INFECTION WITH THIS BACTERIUM, USING WHOLE-GENOME SEQUENCING TO IDENTIFY VIRULENCE POTENTIAL (ABILITY TO INFECT/DAMAGE HOST TISSUES).